FLT3 c.2503G>T, p.Asp835Tyr

Reasoning

PM2 is met at Supporting strength. The exact FLT3 NM_004119.3:c.2503G>T (p.Asp835Tyr) variant was absent from gnomAD v2.1 and gnomAD v4.1, and the queried non-cancer subsets were also absent. This is consistent with an allele frequency of 0 in the queried population datasets, satisfying the generic PM2 Supporting threshold <=0.0001. No population-specific frequency, ancestry enrichment, homozygote, or coverage artifact was reported; the evidence is absence rather than a quantified allele count.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• GNOMAD_V2_1: exact variant absent from gnomAD v2.1; the queried non-cancer subset was also absent.
• GNOMAD_V4_1: exact variant absent from gnomAD v4.1; the queried non-cancer subset was also absent.
• Applied PM2 at Supporting strength using the supplied generic SVI threshold <=0.0001, cited to ACMG/AMP 2015 and ClinGen SVI guidance (PMID:25741868).

Sources

Reasoning

Scope was settled first: NM_004119.3:c.2503G>T is an exonic missense variant, p.(Asp835Tyr), so generic PP3 uses the REVEL path only; SpliceAI is not used for missense PP3/BP4 assessment. The REVEL score is 0.875, which meets the supplied ClinGen SVI moderate PP3 threshold of >=0.773 but is below the strong threshold of >=0.932. The BayesDel score is not used because no generic ACMG-strength calibration is established for BayesDel, and the SpliceAI result is not counted on the missense path.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.).

Evidence considered

• Variant consequence: NM_004119.3:c.2503G>T is an exonic missense variant producing NP_004110.2:p.(Asp835Tyr).
• REVEL score = 0.875 for this variant; this is the applicable predictor path for a missense variant.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, American Journal of Human Genetics, PMID:36413997): PP3 moderate threshold >=0.773; strong threshold >=0.932.
• SpliceAI max delta = 0.149 was not used because generic PP3/BP4 assessment for a missense variant uses REVEL only; BayesDel was not used because no generic ACMG-strength calibration is established.

Sources

Reasoning

The variant is p.Asp835Tyr (p.D835Y), and the reviewed literature reports this same nucleotide/protein event, including Asp835Tyr in 5 of 97 AML cases (PMID:11442493). However, PS1 requires the same amino-acid change to have been established as pathogenic from a different nucleotide change; the reviewed exact-variant records and papers document D835Y but do not document an alternate nucleotide substitution producing Tyr at residue 835. Therefore the generic PS1 requirement is not met.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• PMID:11442493 reports Asp835Tyr (n=5) among seven codon-835 mutations in 97 adult AML cases, but does not report a different nucleotide change encoding Asp835Tyr.
• PMID:15256420 directly studies D835Y as the queried protein event; it does not establish a distinct nucleotide encoding the same amino-acid change.
• The FLT3 VCEP files flt3_itd_hotspot_and_function.md and flt3_oncokb_guidance.md were searched for c.2503G>T, p.Asp835Tyr, and p.D835Y; neither file contains an entry for this variant.

Still missing

• No alternate-nucleotide FLT3 variant producing p.Asp835Tyr was documented in the reviewed sources.

Sources

Reasoning

PMID:11442493 reports the exact FLT3 Asp835Tyr/p.(D835Y) variant in 5 of 97 adults with de novo AML and reports Asp835 mutations absent from 80 normal control DNA samples. Treating those counts as a case-control comparison, I calculated the odds ratio using the Haldane-Anscombe correction for the zero control cell: OR = (5.5 x 80.5)/(92.5 x 0.5) = 9.57; the 95% CI is 0.52-175.81 and includes 1. Therefore the ACMG/AMP PS4 requirement of OR >5.0 with a confidence interval that does not include 1.0 is not satisfied. The control result is reported for Asp835 mutations collectively rather than explicitly for D835Y alone, which further limits specificity.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• PMID:11442493, Results/Table I: Asp835Tyr was reported in 5 of 97 adult de novo AML cases; Asp835 mutations were absent from 80 normal control DNA samples.
• Computed case-control comparison from the reported counts using Haldane-Anscombe correction for the zero control cell: OR = 9.57, 95% CI = 0.52-175.81; the CI includes 1.0.

Still missing

• A control count specific to Asp835Tyr/D835Y, rather than Asp835 mutations collectively, is not reported.

Sources

Reasoning

The exact-variant ClinVar record is Pathogenic but has no assertion criteria and no expert-panel submission; the submissions are from clinical laboratories/OMIM and are not an exact-variant expert-panel classification. Under the specified generic rule, this does not trigger PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 16276 for NM_004119.3:c.2503G>T (p.Asp835Tyr): Pathogenic, no assertion criteria provided, review status zero stars, and no expert-panel submission.

Sources

Reasoning

BA1 is not met. The exact FLT3 NM_004119.3:c.2503G>T (p.Asp835Tyr) variant was absent from gnomAD v2.1 and gnomAD v4.1, including the queried non-cancer subsets, so no population allele frequency approached the >=0.05 BA1 threshold. The records report absence rather than an allele count or frequency; this is still decisively below the stand-alone benign threshold.

Rule

Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• GNOMAD_V2_1: exact variant absent from gnomAD v2.1; the queried non-cancer subset was also absent.
• GNOMAD_V4_1: exact variant absent from gnomAD v4.1; the queried non-cancer subset was also absent.
• Applied the published generic BA1 stand-alone threshold >=0.05 from ACMG/AMP 2015 (PMID:25741868).

Sources

Reasoning

BS1 is not met. The exact variant was absent from gnomAD v2.1 and v4.1, including the queried non-cancer subsets, so there is no observed population allele frequency greater than expected for the disorder. The generic 0.01 default threshold is not reached. Because the observed frequency is zero, a disease-prevalence, penetrance, and allelic-heterogeneity maximum-credible-frequency calculation is not needed to reject BS1 here; no ancestry-specific elevation or founder signal was observed.

Rule

Allele frequency is greater than expected for disorder.

Evidence considered

• GNOMAD_V2_1: exact variant absent from gnomAD v2.1; the queried non-cancer subset was also absent.
• GNOMAD_V4_1: exact variant absent from gnomAD v4.1; the queried non-cancer subset was also absent.
• Applied the published generic BS1 default threshold >=0.01 and considered maximum-credible-frequency principles from Whiffin et al. 2017 (PMID:28518168); no elevated ancestry-specific frequency was present to require a disorder-specific calculation.

Sources

Reasoning

BS3 requires well-established in vitro or in vivo functional studies showing no damaging effect on protein function or splicing. The available variant-specific studies instead show damaging functional effects: D835Y constitutively activates FLT3, produces factor-independent Ba/F3 proliferation and downstream signaling, confers inhibitor resistance, and causes a constitutively active myeloproliferative phenotype in a knock-in mouse model. These findings directly contradict the no-damaging-effect requirement, so BS3 is not met.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• PMID:15256420, Results and Table 1: engineered Ba/F3 cells expressing D835Y showed IL-3-independent proliferation, FLT3 autophosphorylation, STAT5 and ERK activation, and relative MLN518 resistance with cellular IC50 greater than 10 μM.
• PMID:24255108, Introduction/Results and mouse-model methods: the murine Flt3 D838Y knock-in, equivalent to human FLT3 D835Y, constitutively activated FLT3 and produced a myeloproliferative neoplasm phenotype.
• PMID:26108694, Results and Discussion: FLT3-ITD D835V/Y/F mutations caused high resistance to type II inhibitors; the study included Ba/F3 functional assays and structural modeling consistent with an active kinase conformation.

Still missing

• No exact c.2503G>T, p.Asp835Tyr, or p.D835Y entry was found in flt3_itd_hotspot_and_function.md.txt or flt3_oncokb_guidance.md.txt; both declared files provide class-level guidance rather than a row for this variant.

Sources

Reasoning

Scope was settled first: this is an exonic missense variant, so generic BP4 uses REVEL only; SpliceAI cannot substitute for a gray-zone REVEL result. The REVEL score is 0.875, which is not within any benign BP4 range and is above the supplied supporting cutoff of <=0.29, moderate cutoff of <=0.183, and strong cutoff of <=0.016. Therefore BP4 is not met. BayesDel is not used because no generic ACMG-strength calibration is established.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc)

Evidence considered

• Variant consequence: NM_004119.3:c.2503G>T is an exonic missense variant producing NP_004110.2:p.(Asp835Tyr).
• REVEL score = 0.875 for this variant; this is the applicable predictor path for a missense variant.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, American Journal of Human Genetics, PMID:36413997): BP4 supporting threshold <=0.29, moderate threshold <=0.183, and strong threshold <=0.016.
• SpliceAI max delta = 0.149 was not used because generic BP4 assessment for a missense variant uses REVEL only; a clean splice prediction cannot replace a gray-zone REVEL result.

Sources

Reasoning

The exact-variant ClinVar record is Pathogenic, not benign or likely benign, and has no expert-panel classification. No qualifying exact-variant ClinVar expert-panel benign/likely benign assertion is available; therefore BP6 is not triggered.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 16276 for NM_004119.3:c.2503G>T (p.Asp835Tyr): Pathogenic, no assertion criteria provided, review status zero stars, and no expert-panel submission.

Sources

Reasoning

The available evidence does not establish a germline de novo event. The relevant case-series paper reports FLT3 Asp835Tyr in affected AML cases and reports somatic status in patients assessed during remission, but provides no proband parental testing, confirmed maternity and paternity, or documented absence of family history. Therefore PS2 cannot be assessed.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• Abu-Duhier et al. (PMID:11442493) reported Asp835Tyr among adults with de novo AML and described somatic status in patients assessed during remission, but did not report parental testing or family history for the queried variant.
• No case evidence documents confirmed maternity and paternity, germline status in the proband, or a disease-compatible phenotype assessment sufficient for PS2.

Still missing

• Proband-level germline testing is not documented.
• Both maternity and paternity are not documented as confirmed.
• No family-history assessment is documented.

Sources

Reasoning

The available evidence does not establish an assumed germline de novo event. The relevant publication describes the variant in AML and supports somatic occurrence in patients assessed during remission, but does not provide a proband-level de novo observation without parental confirmation. PM6 therefore cannot be assessed.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• Abu-Duhier et al. (PMID:11442493) reported Asp835Tyr in AML and found Asp835 mutations to be somatic in patients assessed during remission; this does not constitute an assumed germline de novo observation.
• No proband-level germline result or de novo clinical observation without parental confirmation is available.

Still missing

• A germline variant observation in the proband is not documented.
• An assumed de novo occurrence without parental confirmation is not documented.
• Parental testing information is absent.

Sources

Reasoning

No affected family members, informative meioses, or co-segregation observations are reported for FLT3 c.2503G>T. The available literature is a somatic AML case series rather than a familial segregation study, so PP1 cannot be assessed.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. ... Note: May be used as stronger evidence with increasing segregation data.

Evidence considered

• Abu-Duhier et al. (PMID:11442493) is an adult AML case series and reports no familial cases, affected-relative testing, or segregation analysis for Asp835Tyr.
• No family-based co-segregation data are available for the queried variant.

Still missing

• No affected relatives with the variant are documented.
• No informative meioses or co-segregation analysis is documented.
• Familial disease and variant testing data are absent.

Sources

Reasoning

The generic PP2 rule requires both a low rate of benign missense variation in the gene and missense variation as a common disease mechanism. The case evidence documents recurrent activating FLT3 missense variation and functional effects for D835Y, but it does not provide the required gene-level benign-missense-rate assessment. The criterion therefore cannot be assessed from the governing evidence without importing an unstated threshold or external metric.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• PMID:11442493 reports recurrent codon-835 FLT3 mutations in AML, and PMID:15256420 reports constitutive activation of FLT3 D835Y in Ba/F3 cells.
• Neither reviewed source supplies the gene-level benign-missense-rate evidence required by PP2.

Still missing

• A validated gene-level assessment showing that FLT3 has a low rate of benign missense variation is unavailable in the supplied evidence.

Sources

Reasoning

The available case evidence does not provide an individual patient phenotype or family history sufficiently specific for a disease with a single genetic etiology. The recorded AML/acute lymphoid leukemia condition labels are ClinVar record context, not a detailed patient phenotype or family history, so PP4 cannot be assessed.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Still missing

• Individual clinical phenotype and family history are not available.

Reasoning

BS2 cannot be assessed from the available evidence. The case contains no documented observation of this variant in a healthy adult individual as a germline heterozygote, homozygote, or hemizygote. The exact variant is represented as a somatic FLT3 cancer variant, and absence from population databases does not establish the healthy-adult observation required by BS2. The applicable inheritance model, expected penetrance, and age of onset for a germline disorder involving this exact variant are also not established.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• GNOMAD_V2_1 and GNOMAD_V4_1 report the exact variant as absent; these records provide no healthy-carrier observation.
• ClinVar variation 16276 describes the exact variant in somatic acute leukemia submissions, not as a documented healthy adult germline observation.

Still missing

• Documented healthy adult germline carrier observation for the exact variant.
• Established inheritance, penetrance, and age-of-onset model for a germline disorder involving this exact variant.

Sources

Reasoning

No affected family members have been evaluated for presence or absence of FLT3 c.2503G>T. The available report is a somatic AML case series and supplies no family non-segregation data; therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• Abu-Duhier et al. (PMID:11442493) reports somatic AML cases but does not describe affected relatives tested for the queried variant.
• There is no documented affected-family-member genotype/phenotype comparison from which lack of segregation could be concluded.

Still missing

• No affected relatives with phenotype and genotype data are documented.
• No family non-segregation observation is documented.
• Potential phenocopy and multiple-variant considerations cannot be evaluated without family data.

Sources

Reasoning

The generic BP1 rule requires a gene for which primarily truncating variants are known to cause disease. The supplied evidence instead documents recurrent activating FLT3 missense substitutions, including D835Y, but does not provide a comprehensive gene-level comparison establishing whether disease-causing FLT3 variants are primarily truncating. The required gene-wide variant-spectrum premise is therefore not assessed.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• PMID:11442493 reports recurrent FLT3 Asp835 missense mutations in AML, and PMID:15256420 demonstrates a functional activating effect for D835Y.
• These variant-specific findings do not establish the gene-wide predominance of truncating disease-causing variants required by BP1.

Still missing

• A comprehensive FLT3 disease-variant spectrum establishing that primarily truncating variants cause disease is unavailable in the supplied evidence.

Sources

Reasoning

BP2 cannot be assessed because no affected-proband or germline family data document this variant in trans with a pathogenic variant in a fully penetrant dominant disorder or in cis with a pathogenic variant; the available reports are somatic leukemia observations and do not provide phase.

Rule

BP2: Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• Abu-Duhier et al. 2001, PMID:11442493: Asp835Tyr was observed in AML cases and reported as somatic in remission-tested patients; no parental testing, offspring testing, partner pathogenic variant, or cis/trans phase was reported.
• Grunwald et al. 2007, PMID:15256420: the study used engineered Ba/F3 cells expressing FLT3 D835Y and explicitly reported no patient-level observations or segregation data for the variant.
• Mizuki et al. 2014, PMID:24255108: the study used a knock-in mouse model of the D835Y-equivalent mutation and reported no human patient cases or segregation data.
• ClinVar variation 16276: exact variant submissions are annotated as somatic leukemia observations without assertion criteria or allele-specific phase information.

Still missing

• No parental, offspring, tumor-normal, or other allele-specific testing establishes cis/trans phase.
• No qualifying pathogenic partner variant is documented in an affected proband.
• The applicable inheritance model and penetrance for a germline FLT3 disorder are not established for this variant.

Sources

Reasoning

No case-level evidence documents that this variant occurred in a patient with an alternate molecular basis for the disease. The available literature and case metadata do not establish a competing molecular diagnosis, so BP5 cannot be assessed.

Rule

Variant found in a case with an alternate molecular basis for disease.

Still missing

• Case-level data on an alternate molecular diagnosis are not available.